The present invention relates to preparations useful for conferring passive or active immunity to the parasite, Cryptosporidium parvum. More specifically, the invention relates to a purified glycoprotein constituent of the parasite; use of the glycoprotein in an immunogenic composition; and monoclonal antibodies that bind a particular epitope disposed on the glycoprotein.
Cryptosporidium parvum is a coccidian parasite that causes intestinal disease in humans as well as economically important food animals including calves, lambs, and goat kids. Healthy, immunocompetent adult humans can be infected, but cryptosporidiosis is particularly serious when it occurs in immunodeficient individuals, including neonates, and those who are immunocompromised as a result of medical treatment, or because of other disease, such as infection by human immunodeficiency virus (HIV).
Among domestic animals, cryptosporidiosis is most frequently reported in calves. The ubiquity of C. parvum in dairy and beef operations throughout the U.S. and its importance as a cause of calf diarrhea are well documented. For example, Anderson et al. in Vet. Med. Sm. Anim. Clin. (June 1981) described well-managed, closed-herd dairies in which C. parvum-related morbidity in 1-2 week old calves approached 100%. We conservatively estimate that the combined treatment costs and decreased production losses incurred by the U.S. cattle industry due to cryptosporidiosis alone now exceed $50,000,000 each year.
C. parvum infection begins when sporozoites released from ingested oocysts invade intestinal epithelial cells. Following attachment of the anterior pole of sporozoites to intestinal epithelium, invasion is associated with host cell membrane evagination around the sporozoite and parasitophorous vacuole formation. Vacuole formation in the apical complex of invading sporozoites is thought to represent discharge of invasion mediators from apical organelles. Following invasion, a feeder organelle forms between the parasite and the host cell cytoplasm and increases the interface surface area markedly. This organelle may function in transport of materials between the host cell and developing trophozoite. Two stages of merogony follow trophozoite development. Type 1 merozoites undergo cyclic replication before developing into type 2 merozoites. Type 2 merozoites subsequently give rise to sexual stages. Fertilization follows and results in the production of oocysts which sporulate at the time of passage in feces. Autoinfective sporozoite and merozoite loops in the life cycle may perpetuate infection in immunocompromised hosts.
Since the first cases of human cryptosporidiosis were reported in 1976, Cryptosporidium has become recognized as a common cause of diarrhea in international travelers, children in day-care centers, livestock handlers, and patients with AIDS or other immune deficiency disorders. Among several recent studies that have addressed the prevalence of C. parvum infection in AIDS patients with diarrhea, one study identified Cryptosporidium as the most common enteropathogen in diarrheic AIDS patients (Laughon et al. Gastroenterol. 94:984 (1988)). Dissemination to extraintestinal sites such as the esophagus, lungs, pancreas and liver has also been shown to occur in immune deficient patients (Soave et al. Rev. Inf. Dis. 8:1012 (1986); Ungar et al. xe2x80x9cCryptosporidiosis in Humansxe2x80x9d pp. 67-75, in J P Dubey, C A Speer and R Fayer (eds.), Cryptosporidiosis of Man and Animals, CRC Press (1990)).
Unlike the other major causes of diarrhea, including infection by E. coli, rotavirus and coronavirus, there are no effective control measures available for cryptosporidiosis. Despite the evaluation of more than 90 drugs, none has been of consistent value and no immunization regimen is presently available to protect against infection by C. parvum. Control of C. parvum infection therefore depends on achieving an adequate immune response. An index of adequate response comprises resistance to reinfection following recovery and short-term disease in immunocompetent hosts. The disease may persist however in immunodeficient hosts.
While cell-mediated immunity is important to naturally occurring resistance to many coccidial species, the evidence suggests that antibody responses can also be manipulated to control infection with sporozoan parasites. For example, hyperimmune bovine serum or colostral whey against whole C. parvum neutralized sporozoite infectivity and partially protected mice and calves against oocyst challenge. Additionally, oral administration of hyperimmune bovine colostrum to persistently infected immunodeficient patients was followed by cessation of diarrhea and oocyst shedding. Further, monoclonal antibodies (mAbs) reactive with C. parvum sporozoite and merozoite surface epitopes neutralized their infectivity and partially protected mice against oocyst challenge.
According to a first aspect of the invention there is provided a monoclonal antibody having the epitope binding specificity of antibody 3E2. In one embodiment, the monoclonal antibody and antibody 3E2 compete with each other for binding to an antigen that is present in a preparation solubilized C. parvum sporozoites. This monoclonal antibody, which competes with antibody 3E2 for antigen binding, stimulates a CSP-like reaction after contacting C. parvum sporozoites. According to a different embodiment, the monoclonal antibody can have an IgM isotype. In a particular case, the monoclonal antibody is antibody 3E2. The invention also provides a hybridoma that secretes the monoclonal antibody 3E2.
According to a second aspect of the invention there is provided a pharmaceutical composition for administration to a mammal, comprising a monoclonal antibody secreted by hybridoma 3E2 and a pharmaceutically acceptable carrier. The invented composition also can include at least one monoclonal antibody other than the monoclonal antibody secreted by hybridoma 3E2. In a preferred embodiment, this other monoclonal antibody has an epitope binding specificity different from the binding specificity of the monoclonal antibody secreted by hybridoma 3E2. The carrier of the pharmaceutical composition can include at least one stabilizing agent which may be a protease inhibitor, a carrier protein or a pH buffering agent. In one embodiment, the carrier optionally comprises colostrum, for example, bovine colostrum.
A third aspect of the invention relates to a method of providing to a mammal passive immunity against C. parvum infection, comprising the step of administering to the mammal a composition comprising antibody 3E2, thereby providing passive immunity. In one embodiment, the composition administered to the mammal comprises a C. parvum neutralizing amount of monoclonal antibody 3E2. According to a different embodiment, the administered composition includes at least one monoclonal antibody other than antibody 3E2 that specifically binds a C. parvum antigen. In a preferred method the composition comprising antibody 3E2 is administered orally in the administering step. In another preferred embodiment of the method, the mammal is a human and in a particularly preferred embodiment the mammal is an immunocompromised human.
A fourth aspect of the invention relates to an isolated circumsporozoite-like antigen of C. parvum which includes a glycoprotein having a molecular weight of 1,400 kDa and which harbors an epitope specifically recognizable by monoclonal antibody 3E2. The isolated antigen can be isolated by a method that includes centrifugation, more particularly, density gradient centrifugation. Alternatively, the isolated antigen can be isolated by methods that involve immunoprecipitation, isoelectric focusing or preparative polyacrylamide gel electrophoresis.
A fifth aspect of the invention relates to an immunogenic composition which includes: (a) a substantially purified C. parvum antigen specifically recognizable by monoclonal antibody 3E2; and (b) a pharmaceutically acceptable carrier. In one embodiment, the C. parvum antigen is a glycoprotein with a molecular weight of approximately 1,400 KDa. In another embodiment, the carrier of the immunogenic composition can include an adjuvant.
A sixth aspect of the invention relates to a method of neutralizing C. parvum infection in a mammal, where the method includes the step of administering to the mammal a composition which includes an antibody having binding specificity for an epitope specifically recognizable by mAb 3E2. The administered composition can include monoclonal antibody 3E2 itself, and may include a C. parvum neutralizing amount of monoclonal antibody 3E2. Alternatively, the administered composition can include at least one monoclonal antibody other than monoclonal antibody 3E2. The administered composition can be administered orally.
Still another aspect of the invention relates to a method of stimulating an anti-C. parvum immune response in an animal. Practice of the invented method involves first obtaining an immunogenic composition that includes a purified C. parvum antigen dispersed in a pharmaceutically acceptable carrier, where the antigen is specifically recognizable by mAb 3E2, and then administering the composition to the animal according to a vaccination protocol. The immunogenic composition used in the invented method optionally can include an adjuvant.
As disclosed herein, the 3E2 monoclonal antibody (mAb) has binding-specificity for a C. parvum surface and apical glycoprotein complex of sporozoites and merozoites, and efficiently neutralizes C. parvum infection in vivo. Interestingly, the epitope recognized by the mAb 3E2 exhibits properties consistent with a carbohydrate/carbohydrate-dependent structure. More particularly, the target epitope, immobilized in a Western blotting format, was sensitive to degradation by glycosidase and degradation by periodate treatment, but resistant to protease digestion in a dot-blotting format. Thus, it is highly unlikely that the epitope could be expressed in recombinant form in a procaryotic host, for example. As will be apparent from the following disclosure, the production and isolation of the 3E2 hybridoma was made possible by the use of purified native C. parvum antigens as immunogens.
The present invention is useful for treating and preventing cryptosporidiosis in all mammals, including human neonates, and also in immunodeficient and certain immunocompetent children and adults. More particularly, the invented composition comprising the mAb 3E2 can be administered to young ruminants, for example, calves, goats and sheep. The invention is useful in these applications because young domestic ruminants serving as food animals are typically housed under conditions where exposure to C. parvum is unavoidable and typically do not possess functionally mature immune systems capable of fighting off infection at the time of exposure to the parasite. Another application of the invention relates to the control of cryptosporidiosis in humans having acquired or congenital immunodeficiencies, including individuals having acquired immunodeficiency syndrome (AIDS) resulting from infection with the human immunodeficiency virus (HIV).
Therapeutic or prophylactic compositions falling within the scope of the invention include preparations containing the mAb 3E2, optionally containing stabilizing agents including protease inhibitors, carrier proteins and pH buffering agents. A convenient source of agents known to stabilize antibodies, particularly when administered by oral route, is colostrum. Colostrum naturally contains antibodies which can, for example, be passed to a neonate. Thus, contemplated formulations for delivery of mAb 3E2 may involve dispersion of the mAb into colostrum prior to administration to a recipient mammal. Notably, experimental results presented herein proved that ascites preparations could be administered orally with strong retention of C. parvum neutralizing activity characteristic of mAb 3E2. This confirmed the utility of mAb 3E2 as an agent having powerful neutralizing activity directed against the parasite, C. parvum. 
As disclosed herein, we have employed competition binding assays to identify mAbs having binding specificities for the same, similar or overlapping epitopes. Results of testing using these assays revealed that mAbs which neutralize C. parvum infection in vivo and which elicit the CSP-like reaction upon contacting sporozoites all have binding specificities that overlap the binding specificity of mAb 3E2. In view of the identity between neutralizing mAbs having the ability to elicit the CSP-like reaction and those having the ability to compete with mAb 3E2 for antigen binding, we concluded that identification of mAbs that compete with mAb 3E2 for antigen binding represents a method of identifying mAbs that can stimulate the CSP-like reaction in C. parvum. Moreover, our results support that any mAb that specifically competes with mAb 3E2 for antigen binding will also have neutralizing activity and be capable of stimulating the CSP-like reaction.
Another aspect of the invention relates to a method of stimulating an anti-C. parvum immune response in an animal using as an immnunogen a purified glycoprotein constituent of C. parvum, wherein the constituent is characterized as a target for binding by mAb 3E2. Immunization with characterized molecules known to be the antigenic targets of a neutralizing humoral immune response, advantageously focuses the immune response against critical epitopes rather than against potentially irrelevant epitopes otherwise found in whole organism preparations. As described below, we have now defined critical epitopes disposed on the surface of C. parvum to which an antibody response must be directed in order to optimize immunoprotection against infection by this organism.
The hybridoma cell line secreting mAb 3E2-A8-B2 (3E2) has been deposited on Mar. 28, 1996 with the American Type Culture Collection Manassas, Va. in compliance with the procedures specified for the deposit of biological materials under the Budapest Treaty. The deposit has been assigned accession number ATCC HB 12075.
The starting point for the development of the invention involved the purification of particular native antigens from C. parvum sporozoites. The C. parvum isolate used in the procedures described herein was obtained from H. Moon (National Animal Disease Center, Ames, Iowa). The C4A1 mAb, described in detail by Mead et al. (J. Parasit. 74:135 (1988)) and Arrowood et al. (Infect. Immun. 57:2283 (1989)), was used in the following procedure as an immunoaffinity purification reagent. Significantly, the purification protocol described below advantageously allowed the isolation of C. parvum protein constituents having posttranslational modifications that would have been absent from, for example, recombinant antigens produced in a heterologous host. General methods useful in immunoaffinity purification procedures can be found in Monoclonal Antibodies: Principles and Practice, 2nd ed. J. W. Goding, Academic Press, London (1986) pp. 219-227, the disclosure of which is hereby incorporated by reference.
Example 1 describes one procedure useful for isolating antigen constituents of C. parvum. 